Project Summary Hybridization Chain Reaction: In Situ Ampli?cation for Biological Imaging Life is orchestrated by programmable biomolecules ? DNA, RNA, and proteins ? interacting within complex biolog- ical circuits. RNA in situ hybridization (RNA-ISH) methods provide biologists with a crucial window into the spatial organization of this circuitry, enabling imaging of mRNA expression in an anatomical context from subcellular to organismal length scales. Due to variability between specimens, examination of detailed spatial relationships requires multiplexed experiments in which multiple target mRNAs are imaged with high resolution within a single biological sample. Using traditional RNA-ISH methods in thick auto?uorescent samples including whole-mount vertebrate embryos, multiplexing is cumbersome or impractical, spatial resolution is frequently compromised by diffusion of reporter molecules, and staining is non-quantitative. The same drawbacks apply using traditional immunohistochemistry (IHC) methods to image protein expression in these challenging samples, while with tradi- tional DNA in situ hybridization (DNA-ISH) methods, it is not currently routine to image single-copy small genomic loci in any sample, much less in vertebrate embryos. These longstanding shortcomings of traditional ISH and IHC methods are a signi?cant impediment to the study of genetic regulatory networks in systems most relevant to human development and disease. In situ ampli?cation based on the mechanism of hybridization chain reaction (HCR) draws on concepts from the emerging discipline of dynamic nucleic acid nanotechnology to achieve three mRNA imaging breakthroughs in whole-mount vertebrate embryos and thick tissue sections: straightforward 5-channel multiplexing, subcellular relative quantitation, and single-molecule resolution and sensitivity. The proposed research will build on these unique capabilities to dramatically advance the robustness, multiplexing, and quantitation capabilities of HCR for RNA-ISH and to extend the bene?ts of multiplexed, quantitative, enzyme-free HCR signal ampli?cation to IHC and DNA-ISH in thick auto?uorescent samples. Major goals are: In situ HCR v3.0: automatic background suppression using cooperative probes for next-generation robust- ness and signal-to-background imaging mRNAs and short RNA targets (miRNAs, mRNA splice junctions, and closely related RNA sequences) in diverse organisms. Next-generation multiplexing (15-plex with simultaneous HCR signal ampli?cation for all targets) and quan- titation (high-?delity mRNA absolute quantitation with subcellular resolution and whole-embryo scale). Next-generation versatility: extend the bene?ts of HCR imaging to protein targets, single-copy small ge- nomic loci, and molecular complexes, enabling compatible multiplexed imaging of all target classes. Realization of these goals would have a broad impact on research in the biological sciences, providing an un- precedented combination of multiplexing, quantitation, resolution, sensitivity, and versatility for the study of genetic regulatory networks in an anatomical context.